Process for preparing 5 hydroxy-tryptamine through new intermediates



PROCESS FOR PREPARING 5 HYDROXY-TRYPTA- MINE THROUGH NEW INTERMEDIATESRomeo Justoni, Milan, and Ralfaele Pessina, Casatenovo Brianza, Italy;said Pessina assignor to Francesco j 'Vismara Societa per Azioni,,Casatenovo Brianza, Italy No Drawing. Original application Feb. '21,1956, so;

. No. 566,776. Divided and this application Feb. 18,

1957, Ser. No; 644,704 I '2 Claims. (Cl. Zak-319) This invention relatesto anew process for preparing the therapeutically activeS-hydtoxy-tryptamine having is the formula:

now discovered a new. process for the preparation of this compoundemploying;- certain; novel intermediates which are illustrated below,which is more advantageous than the processes proposed hereto.

Most of the new intermediates used in our new process thep.benzyloxy-phenylhydrazone of the akjeto adipic acid, having theformula 1 jonr-ongonro ooH o-ooon Nit-ii I I CaHr-CHr-O and thecorresponding I di-alkyl ,esters, tormed withl straighter branched alkylgroups containing up toff carbon atoms. a e j v One of the objects ofthe present invention is to provide a new process for preparingj-hydroxy-tryptamine.

Another object is to provide the above said new'interl mediate compoundsand methods for the production thereof. 1 -T J 1 The starting materialsare well known cheap substances which may be technically manufactured,by easy and ecq nomical procedures, according to methodsfl already described in the chemical literature; they are; p.benzylo ryanilinehydrochloride (which is obtained from p.nit1 'ophenol andbenzylchloride, by passing through pnitroare indole derivatives-whichmay be represented bythe r following general formula? wherer'X :isselected from the group consisting of: Hg

COOH, COOCH ,COOC H and the like alkyl-ester groups; Y being -:COOH or-COOR (R being a straight orbranched alkyl group: containing up to"5carbon atoms); when X is H, Y also being, besides the groups mentionedabove,a member of the class consisting of: -'-'CO--.NH--NH -CON -N=CO,--NH-COOR (R being a straight or branched alkyl group containingup to 5carbon atoms) or --NHCOOCH C H Z being a benzyl group =C H -CH but beingeither C H CN or H, when X is H and Y is a urethane function as NHCOORing). r v

Besides the indole derivatives, represented by the abovesaidgeneralformula, other new substances-which we have discovered asintermediates in the new process for preparing S-hydroxy-tryptathineare: the S-benzyloxytryptamine benzoate of Formula HM and the 5-benzy1-*oxy-tryptamine salicylate'of Formula III-b;

Illa X-H tub x on '(R having the above stated meanof .benzene and itsmethyl homologues' and dioxan, yielding the corresponding indoledeiivatives: e,g. the diphenol-benzylether) anda-carbethoxy-cyclopentanone (which is obtained from the di-ethyl esterof the adipic acid by treatment with sodium). 4

The initial step of our invention consists in the preparation of theabove mentioned a-keto-adipic acid p.ben zyloxy-phenylhydrazone (IV); s1

This new compound is readily-obtained as follows; p.benzyloxy-anilinehydrochloride is diazotised inan ex cess of aqueous hydrochloric acidwith sodium nitrite i and, after addition of sodium acetate, theobtaineddiazo1 compound is reacted with a -carbetoxy-cyclo-pentanone;the precipitated solid product of the couplinghaving the formula vvUHF-CH] oun c are is filtered and treated to ebullit'ion' with aqueoussodium hydroxide; acidification yields the new wketo-adip'ic acidp.benzyloxy-phenylhydrazone of Formula IV(M.Pt;

148-149 C., from diluteethanol). i

By heating in a boiling anhydrous solvent-selected from the classconsisting of benzeneand its methyl homologues and dioxanin thepresenceof a mineral acid the oc-ketO' adipic acidp.benzyloxy-phenylhydrazone yields the new 5-benzyloxy-indole-2carboxyl-3-/3-propionic acid; (For; mula II above,v 'whereX and Y=COOH;Z= CH C H M.P t. 191 192. C., from dilute alcohol). v

In a at 'ctly analogous way,'but by asomewhat difierent procedure, thesame S-benzyloxy-indbl-Z-carboxylffl propionic acid maybe obtainedthrough the corresponding dialkyl esters, For this purpose the newdi-alkyl esters of a-ketoaidipic acid p. benzyloxyphenylhydra zone-whichare easily obtained by treatment of: the a-keto-adipic acidp.benzyloxy-phenylhydrazrine with an esterifying agent selected from theclass consisting; of

methylalcohol, ethylalcohol, and other like alcohol; diazornethane,diazoethane and like diazoaliphatic deriva-. tives containing up to 5carbon atoms (eg. the di methyl ester of the acid of Formula IV; M.Pt.114-115.5 C.,

from methanol) --are treated to ebullition in the presence of a mineralacid inan ,anhydrousalcohol containing up to 5 carbon atoms, as wanes inanotheranhydrous solvent selected from the above mentioned classconsisting s esses M.Pt. 122-4235 C., from ethanol), the analogousdiethyl ester (Formula II, where X and Y=--COOC H Z=CH -C H M.Pt. 108-109-* C.,. from ethanol) and the like. We point out that esterification ofthe u-keto-adipic acid p.benzyloxy-phenylhydrazone and conversion 'intothe corresponding 'indole derivative may be also carried out incontinuation in the same alcoholic solvent in presence of a mineralacid; without isolating the intermediate ester ofthe acid of Formula IV.Alkaline hydrolysis of the said indole di-esters (Formula II, where Xand Y arecarboxy alkyl groups and-Z is benzyl group) furnishesS-benzyloxy-indole-Z-CarboXy-3-/3-propionic acid;

indoledicarboxylic acid is then transformed by thermic decarboxylationinto a benzyloxy-indole monocarboxylic acid (Formula II above where:Y=COOH,

and Z= --,CH -C H which, besides the S-benzylony group, contains in theindole nucleus, only a CHfGHg-QQOI-I side-chain in 3-position. This newcompoundis submitted to a number of'transformations, through a series ofintermediates, in order to convert the carhoxylic-Ihside-chain into av--CH CH --NH side;- chain, and to convert theS-benzyloxy-group into afree S-hydroxyl group.

In practicing. these steps of our invention, S -benZyIoXyindole-2-carboxyl-3-fl-propionic acid is heated preferably at190-230 C.,. alone or in solution and/or suspension in an inerthigh-boiling liquid, selected from the class consisting oftetrahydronaphthalene, decahydrona-phthalene and neutral petroleumderivatives having a boiling point above 190 C. such asparaffin oil;mono-decarboxylation of-the dicarboxylic acid'occurs and S-benzyloxy-indole-S-B-propionit: acid" (Formula II where: Y=COOH, X=H and'Z=CH-C H M. Pt. 163- 165 C., from dilute ethanol) is obtained.

This indole' mono-carboxylic acid is esterified with a low molecularalcohol in order to obtain a corresponding new ester: for example themethyl-ester (Formula II where: Y= C OQGH X=H and Z=CH -C H M.Pt. 98l01C., from methanol), the ethyl ester (Formula II where: Y=COOC H X=H andM.Pt. 62*63: C., fromhexane and other similar-esters with alkylgroups-containing up to 5 carbon atoms.

By boiling with an alcoholichydrazinehydrate solution theso obtainedester is then converted into the corresponding-hydrazide (Formula II,where;

M.Pt. 137-138 C., from dilute ethanol).

By treating the said hydrazide with sodium nitrite and an acid inpresence of water and of a solvent, selected from the group consistingof ether, 'benzene and its methyl homologues' and aliphatic alcoholscontaining 4 or 5 vcarbon atoms, the hithfi iqunknown azide. (FormulaII, where: Y=CQN X=H and Z =..CHg.-.C H is read-. ily obtained. 7

As we have verified this azide-bypyrolysis at 60- 140 C., inaninertsolvent, and subsequent mild hydrolysis of the so formed isocyanate .(FormulaII where: Y NCO, X=H and Z=-CH --C3H with anaqueous mineralacid-may be directly transformed into 5- benzyloxyqtryptamine which, inits, turn, may be converted into' 5 -hydroxy'-tryptamine. However wepoint out that this way of transforming the. above, azide into 5-hydro;ry-tryptanrine furnishesonly a, very poor. yield. On the contrarywe have discovered that it is possible. in the practice of our inventionto convert in ,a high yield. he ai azide t ydrox w ryp min r bett r indirect procedure through certain further intermediat compounds. Theconversion is carried out as follows: The azide is heated at 60-l 40 C.with an alcohol selected from the class consisting of methyl alcohol,ethyl alcohol, other. low molecular. alcohols containing up to 5 carbonatoms and benzyl alcohol. Pyrolysis occurs and they transitorily formed.isocyanate, by; reaction with. the, alcohol: employed, yields anurethane[Formula II where: Xbe na n ,-.Z.=rCHz:- 6H5 Y s a member ot h la s.- cs i 0 N Q G a N L-6 0 235.

NH-COOQIL}. (normal and iso), Nn -cooc' n,

(normal and isomers), NH-COOC H (normal and isomers), NH-COOC I-IgC H lThe constitution of these urethanes is also clearly illustratedby-thefollow v where: A=a1l yl;or arallgyl. group according to the alcoholemployed in its preparation. For example employing methanol themethyl-urethane-i. e. the 8-[3-(5-benzy1 Xy)-indolyl]-ethyl carbamicacid methyl ester-(Formula V where: A=-CH M.Pt. 94-95 C.from benzene bymixing with a small quantity of hexane-) is obtained. Similarly:ethanol; yields the ethylurethane (Formula V. where: A==-.-C' H5; M.Pt.87-88 C;--' from benzene with hexane--);isoamyl alcohol yieldsthecorresponding isoamylurethane (Formula V where:

The urethane is then subjected to a mild catalytic hydrogenolysis in thepresence of a palladium catalyst in an alcohol solution; by removal of=the ben zyl group from the S-benzyloxy function, the correspondingnon-benzylated urethane having a free S-hydroxyl group is formed(Formula II where X and Z =H; Y is a member of the class consisting of:NHC0OCH NH--COOC H NH--COOC H (normal and iso), NH-C0OC H (normal :andisomers), NH=COOC5H11. (normal and isomers), NH COOCH .C H hydrolysis ofthe latter, in an alcohol solution, employing as hydrolysing agent: amineral acid in=the.-presence.-ofwater, gives, by splitting off: the.-carbamic. ester; group, the; desired S-hydroxy-tryptamine. (.Eormulal)which is-isolatedain the fornrof; its. picrate,- or. oxalates, or ofits. well known double saltz 5 -hydroxyatryptamine-creatinine sulfate;

Particularly. when the benzyl=urethane derivative (FormulaMwhere: A=--CH-C H or; Formula'II; where:

is prepared-as intermediate, the catalytic;hydrogenolysis thereofin thepresence of an aqueous alcohol and ;a mineral" acid, furnishesdirectly-a very high yield'of 5-hydroxy-tryptamine,

Again we have found'that the conversion'of any intermediateS-benzyloxy-urethane (Formula V) into 5-hydrtnqr.-try ptamine may beobtained by carrying. out the same reactions-hydrogenolysis andhydrolysis-in an inverted order, i.e. firstly hydrolysis and thenhydrogenolysis. In this case hydrolysis of the urethane in the Apresence of a mineral acid yields S-benzyloxy-tryptamine [benzoate orsalycilate of -benz yloxy-tryptamine (Illa and IIIb], in an alcoholsolution ives a solution con taining the corresponding salts of5-hydroxy-tryptamine, from which the indole base may be isolated in theform of its picrate. In order that the'inventionmay be well understoodthe following examples are given by way of illustrationonly: t I r i 1Example] (a) 100 g. of paminO-phenQI-benzyI ether hydrochlo ride in 120cc. of water and 76 cc. of concentrated hydrochloric acid are diazotised.with 32.3 g. of sodium nitrite dissolved in 100 cc. of water. 120 g. ofsodium acetate 'are added and then, With'co'ntinuous agitation at'0-"C.,

the solution is coupled with 66.5 g. of ,a-carbetoxy-cyclopentanone.Agitation is continued for three hours allow ing the temperature toriseto 20 C. The formed solid product is filtered, Washed with water, mixedwith 1200 'cc. of a 5% sodium hydroxide solution and heatedon steam bathfor three hours. The warm solutionis 'n'eti tralised to pH 7.5 withdilutefhydrochloric acid and filjtered with charcoal; after coolingthea-k'eto-adipic acid pbenzyloXy-phenylhydrazorie is precipitatedivithhydro chloric acid; the yield of the filtered, washed and dried productis 128 g.: yellow crystallinepovvderflM. Pt. 146 148C. (A samplerecrystallised from 50% ethanol shows M. Pt. 148-l49 c.; thedetermination in N ontent is in agreement with'the formula C H bO N2:calc. 7.86; N found 7.98.) j l f I (b) A solution of 125 g; of the aboveet-keto-adipic acid p-benzyloxy-phenylhydrazone in 1250 cc. of dioxancontaining 5% of dry hydrogen chloride is refluxed over a period oftwenty minutes. After cooling the formed ammonium chloride. is filteredb'y suction and washed with a little of dioxan. The clear filtrate isadjusted .to pH 8 by adding a 10% aqueous sodium carbonate solu- 'tion.The solvent is then distilled at -30mm. pressure so obtaining about300-400 00'. of a concentrated residual solution which, afterneutralisation to pH about 7.2, is decolorised with charcoal andfiltered. The filtrate is made acid to Congo red with hydrochloric acidthus pre cipitating the crude S-benzyloxy-indole2-carboxyl 3-[3-propionic acid which is collected on a Biichner funnel, washed withwater until the washings are neutral to litmus and dried on steam bath.The yield of the crude product melting at about 171-l7 3 C. is 103g.After repeated crystallisations from aqueous ethanol (1&1) 80 g. ofpure, white 5-benzyloxy-indole-2-carboxyl-3-/3 pro picnic acid meltingat l88-190 C. are obtained. (An analytical sample shows M. Pt. 19ll92C.; determination of N content gives values according to the formulaC19H1'1O5NI N calc. 4.12; N found.4;06.)' i

This step (b) may be carried out also employing instead of dioxan thebenzene or toluene as solvent for the indole cyclisation. In this case.howeverthe yields are lower. p (0) Four 20 g. portions of5benzyloxy4indole-2-carboxyl-3-fl-propionic acid, suspended in four.200cc. porjtions of paraffin oil, are heated at ca. 210 C.'in an oilbath; melting and decarboxylation occurs. The heating is continued forone and one-ha1f hours. After coolin'g'at ca. 60' C., the mixture isextracted with 300 cc; of aqueous 10% sodium carbonate solution: thepHof the obtained brown solution is then adjusted to about 7.2 andthe'solution filtered with charcoal. from separated tar. Byacidification with dilute hydrochloric acid, 62 g. of crude5-benzyloxy-indole-3-B-propionic .acid, melting at -150-151 C. withsintering at 118 C. separates off. This productrecrystallised from theminimal amount of 50% aqueous ethanol melts at l53156 C.; yield 42.8 g.(61% of the theoretical amount). (An analytical sample twicerecrystallised from 50% ethanol melts at 163- 165 C. and the nitrogendetermination is ingood agreement with formula C H O N: N found 4.79.)

-"'(d)' Ausoluti'on of 42 g. of the aboveS-benzyldXyindole-S-fi-propioni: -aoid' in 420 cc; of absolute metha--nol, containing 3% ofdry hydrogen chloride, is refluxed fora period oftwo hou'rs.- After cooling, the mixture is poured into a solution of 43g. of sodium bicarbonate in900 ce. of water. The separated crystallinebrownish s'olid iscollec ted on a filter and washed with'water giving43.3 g. of methyl-5-benzyloxy-indole-3-;8-propionate: ML Pt. 98-99 C.(An analytical .samplerecry'stallised from methanol shows M.Pt. 100-101C: for C H O N; N calc: 4.53; N found 4.37.). (e) A mixture of 43 g. ofthe above methyl-5-benzyloxy-indole-S-B-propionate, 1160. cc. of ethanoland ,63 cc; of hydrazine hydrate is heated under reflux for-one hour.After removal under reduced pressureof .most' of the solvent, theresidual solution is diluted-with an equal volume of water, and theprecipitated crysta'lline solid; is filtered and Washed with'vvater. jThe M.Pt. of thecrude hydrazide is 133-135 C. Aftercrystallisation from70% ethanol, pure 5-benzyloxy-indole-3-/3-propionhydrazide is obtainedin an amount of -'40.8 g. (96% of-the theoreticalamount)! M.Pt. 134-l36C. (An analytical sample, recrystallised from water,- melts at137-138-C.: for C H' O N i N 'cal'c. 13.59, N %-found 13.70.) a

(f) To a solution of 40g. .of the above hydrazide in cc. ofacetic.acid,'510 cc. of water. chilled to 0 C. and 330 cc. of benzeneare added with stirring. The resultingmixture is then treated with 100cc. of-a, 10% aqueous sodium nitrite solution,stirringbeingcontinuedfor; five minutes more. After V- separating the benzene layer, theaqueous phase is extracted twice with 330 cc. portions .of benzene. Thetwo 0.-.C. Ecooledcombined benz'ene extracts, are washedfirstlywith acooled dilute sodium. bica'rbonate aqueous solutionandthen with cooledwater until neutral anddried over finely powdered anhydroussodiumsulfate. (A .sample.15' cc.of the filteredbenzene solution,- ,evaporatedto dryness under reduced pressure atroomtemperature, :givesa yellowcrystalline residue of crude 5-benzyloxyeindole-3epepro pionazide whichbegins to decompose at 45ff CZ) (g) The above benzene, solution.isaddeddropwiseto 3300 cc. of boiling anhydrous methanol... Continuousdistillation of the azeot-ropic mixture of methanol and benzene occurs.v I .When all the. benzene had been added and distilled, theresidualmethanol solution is refluxed forfone hour and then evaporated underreducedpressur to dryness thus ,obtaining a brownish oil.v 'Aconcentrated benzene solution of this oil is poured'overffZO g. of.aluminum oxide andthe formed ,6-[3-(55beuzyloxy)-indolyl]-ethyl carbamicacid methyl ester is completely eluted by Walsh.- ing the aluminum oxidewith benzene. Evaporation of benzene solution at reduced pressureitoasmall volume (about cc.) and dilution with 15 cqocf hexane gives 27 g.of the methylurethane .as white needles M.Pt. 90- 93 C. The motherliquors area'gaintreated with aluminium oxide so obtaining a furtheramount of the same product." Total yield 31.5 g. (An analyticalsampletwice recrystallised from benzene-hexane melts at 94-. 95 C.: thenitrogen content is in good agreement with the formula C H O N Ncalc...8.64, N' found 8.72.). L lf, (h) A solution of 31 .g. of theabove methylurethane in 1000 cc. of methanol is treated with hydrogenwith continuous stirring at five' atmgpressurefioom tem peratureiabout25 C.) in presence of 15 g. of a 10% palladium on. carbon catalyst untilno more, hydrogen was adsorbed (about five hours). The filteredmethanolic solution contains fi-l 3-(5-hydroxy) -indolyl]-ethyl carbamicacid methyl ester. It was then evaporated un' der reduced pressure to avolume of about 400cc. 1' (That the debenzylation is accomplished 'isproved by the fact that a sample of this solution treatedinan alkalinemedium. with an aqueous solution of diazotisal sulphanilicacid givesared colour so confirming-the presence of a substance having a freephenolickgroup.)

' The crude methanolic solution containing theJfl-lB-(S-hydroxy)-indolyll-ethyl carbamic'acid methyl ester is IQ fluxed with 150.cc. of dilute hydrochloric acid (1:1) for 30 minutes to hydrolyse thecarbamic ester. The green ish solution, after addition of 9 g. ofcrystallised sodium acetate, is neutralised to Congo red with sodiumbicarhonate andfiltered from the separated sodium chloride. l (i)20.8,g. of picric acid are added to the filtrate, and most of thealcohol is evaporated under reduced pressure. The residual orange-redmixture is .diluted to about 400 cc. with water, warmed to 50-65" C. andfiltered with charcoal: by cooling S-hydroxy-tryptamine picrateseparates oil as red needles M.Pt. l85l87 C. withdec; afterconcentration of :the mother liquors under reduced pressure a furtheramount of the same picrate is obtained; (A sample recrystallised fromwater melts at 196-197" C. and a mixed melting pomt with an authenticsample of 5-hydroxy-tryptamine picrate givesno depression.)

Example 2 (a) a-K'eto-adipic acid p.benzyloxy-phenylhydrazon is preparedas described in the step (a) of Example .1. (b) 128 g. of this productare esterified employing an excess of a; diazomethane methylene chloridesolution; elimination ot the solvent and of diazomethane' excess gives135 g. of the dimethyl-ester in form of yellow crystals. An analyticalsample recrystallized from methanol shows M.Pt. l=14-1l5.5 C. and thedetermination .of N content is in agreement with the constitution ofdimethyl-a-keto-adipate-p.benzyloxy-phenylhydrazone (for C H O N N calc.7.29; N found 7.18). I

134 g. of the above crude dimethyl-ester are suspended in 1660 cc. ofabsolute methanol containing 10% of dry hydrogen chloride and refluxedfor'twenty minutes. After cooling, the reaction mixture is graduallypoured into a cooled solution of 400g. of sodium bicarbonate in 6000 cc.of water. The separated solid brown product is filtered, washed withwater and crystallised from ethanol so obtaining 96.5 g. of whitecrystals, M.Pt. 120-121 C., of dimethyl-S-benzyloxy-indole-2-carboxy-3B-propionate. j The mother. liquors are evaporated to dryness atreducedpressure; the dried residue is again taken up with 350 cc. of absolutemethanol containing 10% of dry hydrogenchloride and again. refluxed fortwenty minutes. By repeating the above. treatment the reaction mixturegives, after crystallisation, further 122g. of the same diester M.Pt.118-120" C. Chromatography on aluminum oxide of a concentrated benzenesolution of the residue of the evaporated mother liquors, by elutionwith hexane and hexane-benzene (1:1) followed by crystallisation fromethanol, gives further 4.3 g. ofdimethyl-S-benzyloxyindole-2-carboxy-3-B-propionate, M.Pt. 120-121 C.

In such a way the yield arises to .113 g. (89% of the theoreticalamount). An analytical sample twice -re-. crystallised from ethanolmelts at 122-1235 -C,.; the analytical values for N are in agreementwith the formula CgiHgiOggNKN calc. 3.81; N found 3.91). Thisdi-methyl-ester is hydrolysed to the corresponding 5benzyloxydndole-2-carboxyl-3 5 propionic acid as tollows: to a solutionof 112g. of di-methyl-S-benzyloxyindole-2-carboxyl-3 B-propionate in2250 cc. of ethanol at 40 (3., 918.5 g. of sodium hydroxide dissolvedin.l64 cc. of waterare gradually added with stirring. The mixture isthen allowed to stand during 12 hours at room temperature (about 25 C.).After cooling at C., the sodium salt, which separates off, is filteredon a Biichner funnel, washed with ethanol and dissolved in the strictlynecessary quantity of water. The aqueous solution decolorised withcharcoal is acidified with dilute hydrochloricaoid and the precipitatedindole-dicarboxylic acid is collectedon a Biichner .funnel, washedthoroughly with water and dried: yield 95 g. of white crystals M.Pt.1901191 C. (92% of the .theoreticalamount). .(c) Five 19 g. port-ions ofS-benzyloxy-indole-Q-carboxyl-3-fl-Ipropionic acid are heated at.215-225" an oil bath; melting and decarboxylation occurs. The heatingis continued for one hour. After cooling, all the obtained brownishsolids are taken up with warm 70% aqueous ethanol and the solutionfiltered with charcoal. Dilution with water to bring the alcohol to 50%and cooling causes the separation of 73 g. of crudeS-benzyloxy-indole-3-B-propionic acid. This product recrystallised fromthe minimal amount of 50% aqueous ethanol melts at 153-156 0.; yield49.5 g.

(d) (e) (f) (g) This 5-benzyloxy-indole-3-,B-propionic acid (49 g.) isconverted into fl-[3-(5-benzyloxy)-indolyll-ethyl carbamic acidmethyl-ester (37 g.) by passing through the steps (d), (e),;(f) and (g)of Example 1.

(h) The handling of this step for transforming the said methylurethaneinto S-hydroxy-tryptamine is inverted with respect to corresponding stepas described in Example 1: the procedure is as follows: firstlyhydrolysis is carried out by treating a solution of 37 g. of themethylurethane in 520cc. of ethanol with 220 cc. of 6 N hydrochloricacid overnight at 4050 (3.: hydrolysis of the carbamic ester groupoccurs. To the so obtained solution containing 5- benzyIoxy-tryptaminehydrochloride, 97.8 g. of sodium bicarbonate are added to neutralise theexcess of hydrochlonic acid. After filtration of .the separated sodiumchloride, 18.5 g. of sodium salicylate are added with stirring and theethanol is evaporated under reduced pressure up to aresidual volume ofabout 250 cc. By cooling crystallisation occurs; the collectedS-benzyloxy-tryptamine salicylate, washed with a little water and dried,forms yellow crystalline needles melting at l67-169 C.; yield 18.5 g. (Awater recrystallised analytical sample in form of white needles showsM.Pt. l74175 C.: the nitrogen determination is in good agreement withthe formula C H ON .C H 0 or C H O N N calc. 6.92;N found 7.01.)

A solution of 18 g. of the above S-benzyloxy-tryptamine salicylate in540 cc. of ethanol is treated with hydrogen at five atm. pressure withcontinuous stirring (room temperature; about 25 C.) in presence of 9 g.of 10% palladium on carbon catalyst until no more hydrogen is adsorbed.

(i) From the filtered alcoholic solution containing 5-hydroxy-tryptamine.salicylate, the indole base is separated asvpicrateby adding 10.3 g. of picnic acid as already is described at thestep (i) of Example 1. By evaporating the orange-red solution to a smallvolumecrystallisation begins. The filtered S-hydroxy-tryptamine picrateis washed with ether to eliminate any trace of salicylic acid andrecrystallised from water; it is so obtained in form of red needlesmelting at 196-197 also when mixed with an authenticsample.

(I) If desired the S-hydroxy-tryptamine may be also separated in theform of its hydrogen oxalate. For this purpose 5 g. ofS-hydroxy-tryptamine picrate are finely suspended in 25 cc. of Nhydrochloric acid. This suspension is long pounded witha rod of glassand repeatedly extracted with ether so as climate picnic acid; in thisway a limpid aqueous solution of S-hydroxy-tryptamine hydrochloride isobtained. The ethereal solution is then washed with 10 cc. of Nhydrochloric acid. The clear aqueous solution (cc. 35) is at firstneutralised to Congo red with sodium bicarbonate and then adjusted to pH7.8-7.9 by addition of sodium carbonate. The liquor is extractedrepeatedly with butanol, washing butanolic extracts with a sodiumchloride solution adjusted to pH 7.9 with potassium carbonate. To thebutanolic solution, previously dried over sodium sulfate, 3 g. of oxalicacid in 25 cc. of ethanol are added and the butanol is then completelyevaporated under reduced pressure. The residue is taken up with a smallwarm absolute ethanol and the alcoholic solution filtered with charcoal;by cooling and addition of dry ether the S-hydroxy-tryptamine hydrogenoxalate separates off as pale bufl microcrystals. A furtherrecrystallisation from ethanol-ether gives a colourless product, M.Pt.195197 C. (Analysis: for C H O N N calc. 10.52; N found 10.35.)

Example 3 (a) By the same procedure'as described in the step (a) ofExample 1 wketo-adipic acid p.benzyloxyphenyl-' hydrazone is prepared.(b)' 125 g. of this compound is suspended in 500 cc. of anhydrous etherand treated with anexcess of a diazoethane methylene chloridesolution(prepared in its turn treating 300 gjof crude moistened 80% ni-trosoethylurea obtained from propionamide according to' the method of A. W.Hofmann, Br. 15, 754, 1882, and Werner, Soc. 115, 1100, 1919-With aconcentrated aqueous potassium hydroxide solution in the presence of3500 cc. of methylene chloride, analogously to what is described inHelv. him. Aota 24, 1474, 1941, with reference tothe like nitrosomethylurea and diazomethane). After 24' hours the excess of diazoethaneis destroyed by shaking with dilute hydrochloric acid, and the methylenechloride evaporated; The crude viscous diethyl-ester is dissolved in3000 cc. of benzene; 1000 cc. of the solvent are distilled to eliminateazeotropically any moisture and into the boiling solution dry hydrogenchloride is bubbled oil er a period of one hour. The cooled reactionmixture is poured into an excess of an aqueous 5% sodium-bicarbonatesolution and the separated benzene layer is con- 'centrated to aresidual volume of about 300 cc. This resiis adsorbed on 2500, g. ofaluminium oxide. Elution with hexane and hexane-benzene (1:1) givesfractions, which after crystallisation from ethanol shows M PL 108-'109" C.: the determination of the nitrogen content is in agreementwiththe constitution of diethyl-S-benzyloxyjndole-2-carboxy-3rfi-propionate(for C H O N N calc. 3.54;N found 3.70). The same procedure isfollowedusing toluene and xylene (o, m, p. mixture) as "solvent for theindole cyclisation. The same diethyl-ester is obtained. v This diethylester is hydrolysed -in analogous way as described in the step (b) ofExample 2 for the corresponding di-methyl ester. 7 ('c) (d) (e) (f) (g)The so obtained S-benzyloxy-in- 'do1e-2-carboxyl-3-,B-propionic acid isthen converted in p 3.-(. ,-benzy1oxy)-indolyll-ethyl carbamic acidmethyl ester according to procedure alreadydescribed at the steps (c),(d), (e), (f) and (g) of Example 1.

,(h) A 20 g. portion of the so obtainedfl-[B-(S-benzyloxy)-indolyll-ethyl carbamic acid methyl ester is hydro-'lysed in a perfectly analogous way as described in the step (h) ofExample 2. To the neutralised hydroethanolic solution filtered fromsodium chloride, 9 g. of sodium 'bjenzoate'are added. By conce'ntrationunder reduced pressure, 5-benzyloxy-tryptamine benzoate as yellowneedles, M.Pt.'148149C. is obtained. (An analytical sample twicerecrystallised from Water melts at 153154 'C. and the nitrogen contentdetermination is in good agreement with the formula "-c H ON C l-l o orC H O N N calc. 7.22, N found 7.40.)

Analogously as in Example 2 at the end of the step (h) ,analcoholicsolution of 9 g. i-benzyloxy-tryptamine benzoate is treated withhydrogen in presence of 10% palladium on carbon catalyst.

(0 By addition ofpicric acid (5.15 g.) and, concentra- :tion oftheorange-red solution, the 5 hydroxytryptamine pic'rate, is separated byhandlng as described at the end ofthe'step (i) of Example 2.

' (I). If desired, from the S-hydroxy-tryptamine picrate may.be preparedthe S-hydroxy-tryptamine oxalate, ac-

cording to the directions described in Example 2 at step (I) for thecorresponding hydrogen'oxalate, with the sole difference that'the oxalicacid employed in calculated "amount for obtaining the neutrum oxalate.'oThe S-hy- 'droxy-tryptamine oxalatemelts at .193-195 ('dec.) after6115" crystallisation from ethanol. (Analysis: for .2

CwHmONg, C2H204 N Cale. N found is prepared as described in the step (a)of Example 1.

(b) g. of this dried product are at once treated with 1500 cc. ofabsolute ethanol containing 10% of dry hydrogen chloride and the mixtureallowed to stand at room temperaturefor 24 hours is then refluxed fortwenty minutes. The transitorily formed diethyl-ester is so directlytransformed into the diethyl-S-benzyloxy-indole-2carboxyl-3-B-propionate. This product is isolated by pouring the cooledreaction mixture into a cooled diluted solution of sodium bicarbonateand by filtering the solid slightly pitchy precipitate. The so obtaineddiethyl-ester after crystallisation from ethanol shows M.Pt. l08-109 andis identical to the product obtained as said in the precedingExample 3at step (b).

The step of esterification and indole cyclisat-ion of a-keto-adipic acidp.benzyloxy-phenylhydrazone may be carried out in absolute methanolinstead of absolute ethanol. In this case-by same procedure as above re;ported the dimethyl-5-benzyloxy-indole-2-carboxyl-3-13- propionate isobtained. This product recrystallised from ethanol melts at 120121 andis identical to the dimethyl-ester obtained asjsaid in the step (b) ofExample 2.

The indole dicarboxylic ester (methyl or' ethyl) is bydrolysed asdescribed at step (b) of Example 2 to the corresponding5-benzyloxy-indole-Z-carboxyl-3 fl propionic acid.

1 (c) (d) (e) 80 g. of this indole dicarboxylic acid are then convertedinto 5-benzyloxy-indole-3-B-propionhydrazide by passing through thesteps (0), (d) and (e) of Example 1. l

(f) The 5-benzyloxy-indole-3-p-propionhydrazide so obtained (40 g.) isconverted into the corresponding azide as reported in the step (f) ofExample 1, with the sole dilference that ether is employed in place ofbenzene.

(g) The ethereal solution of azide, about 650 cc., dried over powderedanhydrous sodium sulfate is added dropwise to 1300 cc. of boilinganhydrous ethanol in such a rate that ether distills off.

When all the ethereal solution has been added and distilled, theresidual ethanolic solution is refluxed for one hour and then evaporatedto dryness under reduced pressure thusobtaining a brownish oil. Aconcentrated benzene solution of this oilis poured over 65 g. ofaluminium oxide and the [i-[3-(S-benzyloxy)-indolyl]-ethyl carbamic acidethyl ester is completely eluted by washing the aluminium oxide withbenzene. I

Evaporation of benzene solution to a small volume (about cc.) anddilution with'13 cc. of hexane gives about 28 g. of the said ethylurethane. The mother liquors were again treated with aluminium oxide soobtaining a further amount of the same product. Total yield 32.2 g.:M.Pt. 86-87 C. (An analytical sample twice recrystallised from benzenehexane melts at 87-88, C. The nitrogen determination is according to theformula C20H2203N2: N calc. 8.28, N fOUlld (h) A solution of 30 g. ofthe above ethylurethane in 1050 cc. of ethanol is treated with hydrogenwith continuous stirring at five atm. pressure (room temperature:"about, 25 C.) in the presence of 10% palladium carbon catalyst until nomore hydrogen is adsorbed (about five hours). The filtered solution,which contains {i-[3-(5-hydroxy) -indolyll-ethyl carbamic acid ethylester, is then evaporated under a reduced pressure to a volume of about450 cc.

t The crude solution of theso formed non-benzylated ethylurethaneisrefluxed with cc. of dilute hydrochlor'ic acid (1:1) for 30 minutes tohydrolyse the carbamic ester group. The green solution, after additionof '9 g. of crystallised sodium acetate, is neutralised to Congo ed withsodium bicarbonate and filteredfrom the separated sodium chloride.

1 1 (i) 20.2 g. of picric acid are added to the filtrate, and most ofthe alcohol is evaporated under reduced pressure. Theresidual-orange-red mixture is diluted to about 450 cc. with'water,warmed to 60-65 and filtered with For of 2 N sulfuric acid are added.The resulting suspension is heated to boiling and then cooled at C.S-hydroxy- 'tryptamine-creatinine-sulfate separates off as a whitecrystalline precipitate. The product is filtered on a Biichner funnel,washed with alcohol and dried. Yield 7.9 g.: M.Pt. 210-211 C. with dec.(A' water recrystallised sample melts at 213-214 C.)

Example (a) p -Keto-adipic acid p. benzyloxy-phenyl-hydrazone isprepared as described in the step (a) of Example 1.

(b) 128 g. of this product are refluxed for twenty minutes with 1500 cc.of a solution of dry hydrogen chloride in sec. butyl carbinol (activeamyl-alcohol of fermentation). The cooled reaction mixture, Washedfirstly with an excess of 5% aqueous sodium bicarbonate solution andthen with water with neutral, is dried and evaporated to dryness underreduced pressure. Chromatography on aluminium oxide of a concentratedbenzene solution of the residue, gives, by elution with hexane andhexane-benzene 1:1), thedi-Z-methyl-butyl-S-benzyloxy-indole-Z-carboxy-3-fi-propionate in theform of a viscous colourless oil. Saponification of this ester with aWarm alcoholic solution of sodium hydroxide gives 90 g. of5-benzyloxy-indole-2-carboxyl-3- 8-propionic acid.

-,(c) This indole dicarboxylic acid (90 g.) is refluxed with. 720 cc. ofboiling tetrahydronaphthalene for seven hours, until the evolution ofcarbon dioxide has ceased. After cooling, the separated crystallineproduct is filtered and dried: 66 g. of5-benzy1oxy-indole-3-;8-propionic acid, melting at l5 3l57 C., areobtained. Concentration of the mother liquors under reduced pressuregives a second crop of 6.4 g. melting at 151-155 C. The total yield ofS-benzyloxy-indole-3-B-propionic acid amounts in this way to about 92%of the theoretical amount; the product is satisfactory for the nextstep. (A mixed melting point of a sample recrystallised from aqueousethanol with a pure sample of the indole monocarboxylic acid gives nodepression.) By extraction of the residual mother liquor with a diluteaqueous sodium bicarbonate solution and following acidification thereof,5.5 g. of a mixture of monoand not changed bi-carboxylic acid melting at1-7l-176 C. are obtained. This third crop may be recycled so giving afurther small amount of S-benzyloxy indole-3-fl-propionic acid.

The same procedure has been experienced using decahydronaphthalene assolvent for the decarboxylation: a similarly good yield of the sameindole-mono-carboxylic acid is obtained.

(d) A solution of 72 g. of 5-benzyloxy-indole-3-fi-propionic acid in 720cc. of isoamyl alcohol containing 3% of dry hydrogen chloride isrefluxed over a period of two hours. After cooling, the mixture is firstwashed with an excess of a dilute aqueous sodium bicarbonate solutionand then with water until neutral thus obtaining an isoccz-of thissolution is dried over magnesium sulfate and then evaporated to drynessin vacuo. A concentrated benzene solution of theresidual brownish oil ischromatographed on 50 g. of aluminium oxide. After complete elution withhexane, benzene and a mixture consisting of equal volumes of benzene andether, followed by evaporation of eluates under reduced pressure, theisoamyl ester is obtained as a pale yellow glass: 3.2 g. The latter isdissolved in 70 cc. of ethanol at 40 C. and hydrolysed by adding asolution of 2.8 g. of sodium hydroxide in 7 cc. of water. After standingat room temperature (about 25 C.) during 24 hours, the mixture is cooledto 0" C. and theseparated sodium salt, filtered on a Biichner funnel andwashed with ethanol. An aqueous concentrated solution of the sodiumsalt, is acidified with dilute hydrochloric acid and the precipitatefiltered, washed thoroughly with water and dried. The productabout 2.1g. of white crystals melting at 161162 C.is recognised as5-benzyloxy-indole-3-18-propionic acid by a mixed melting point with anauthentic sample.)

(e) The above isoamyl alcohol solution containing the main fraction ofisoamyl ester is refluxed with cc. of hydrazine hydrate for one hour.After cooling, the solution containing the5-benzyloxy-indole-3-,6-propionhydrazide is transferred to a separatingfunnel and repeatedly washed with water and, finally, with watercontaining a little hydrochloric acid to eliminate the excess ofhydrazine. (A 35 cc. sample of the dried isoamy-alcohol solution afterevaporation in vacuo gives a crystalline residue which, aftercrystallisation from dilute ethanol, melts at -136 C. and gives nodepression when mixed with an authentic sample of 5-benzyloxy-indole-3-fl-propionhydrazide.)

(f) To the above isoamyl alcohol solution of the hydrazide a solution of18 g. of sodium nitrite in cc. of water is added. After cooling to 0 C.and stirring 22.2 cc. of concentrated hydrochloric acid diluted with 50cc. of water are dropped in the mixture. After standing for five minutesthe isoamyl alcohol layer is separated and the aqueous phase extractedwith 350 cc. of isoamyl alcohol. The combined extracts, washed firstlywith a dilute aqueous sodium bicarbonate solution and then with wateruntil neutral, are dried over anhydrous potassium carbonate. 7

(g). The filtered isoamyl alcohol solution, containing the5-benzyloxy-indole-3-fl-propionazide, is refluxed for one hour in around-bottomed flask, employing an apparatus equipped in such a way thatthe isoamyl alcohol refluxing in the flask was dried by contact withphosphoric anhydride. Formation of the isoamyl urethane occurs. (A 35cc. sampleof the obtained solution is evaporated to dryness underreduced pressure. A concentrated benzene solution of the residualbrownish oil is poured into 10 g. ofaluminium oxide. Elution withbenzene and evaporation to dryness of the eluates gives the,B-[3-(5-benzyloxy) -indolyl]-ethyl carbamic acid isoamyl ester as apale yellow glass which does not crystalrse.) (h) The above isoamylalcohol solution containing the isoamyl urethane is treated withhydrogen under continuous stirring at five atm. pressure (roomtemperature: about 25 C.) in presence of 28' g. of 10% palladium oncarbon catalyst until no more hydrogen is adsorbed (about five hours).The filtered isoamyl alcohol solution contains,3-[3-(5-hydroxy)-indolyl]-ethyl carbamic acid isoamyl ester. It is thenevaporated to dryness under reduced pressure.

The residue is taken up with 720 cc. of ethanol and after the additionof 120 cc. of water and 120 cc. of concentrated hydrochloric acid isrefluxed for 30 minutes in order to hydrolyse the carbamic ester group.The greenish solution, after addition of 14.4 g. of crystallised sodiumacetate, is neutralised, to Congo red with sodium bicarbonate andfiltered from the separated sodium chloride. 7 7 7 a a (1') 36 g. ofpiciic acid are added to the filtrate and most of the alcohol isevaporated under reduced pres sure. Water warmed to 65 C. is added'to avolume of about 55000. The warm solution is filtered with charcoalz bycooling 5-hydroxy-tiyptamine picrate separates o'fi'as' red needlesM.Pt. 185-1879 with dec.: after 'concentration of the mother liquorsunder reduced pressure a further amount of the same picrate is obtained.(A sample recrystallised from water melts at 195196 C. and a mixedmelting point with a pure sample of 5-hydroxytryptamine picrate gives nodepression.)

Example 6 (a) (b) 5 benzyloxy-indole-2-carboxyl-3-p-propionic acidis-prepared as described in the steps (a) and (b) of Example 1.

(c) 80 g. of this indole dicarboxylic acid are converted into5-benzyloxy-indole-3-fl-propionic acid (64 g.) according the step (c) ofExample 5.

(d) A solution of 64 g. of the above crude indolemonocarboxylic acid in640 cc. of absolute ethanol containing 3% of dry hydrogen chloride isrefluxed for two hours. After cooling the mixture is poured into asolution of 65 g. of sodium bicarbonate in 1300 cc. of water thusobtaining 65.5 g. of the crude ethyl-5-benzyloxy-indole-3-18- propionatewhich after crystallisation from hexane melts at 6263 C. and is in goodagreement with the formula C H N:N calc. 4.33, N found 4.48.

(e) A mixture of 65 g. of the above ethyl ester, 1750 cc. of ethanol and93 cc. of hydrazine hydrate is refluxed for one hour. By treating thesolution in the same way as described in the step (e) of Example 1 forthe transformation of the corresponding methyl ester into the hydrazide,60.5 g. of crude -benzyloxy-indole-3- 3-propionhydrazide, M.Pt. l33134C., are obtained. (The mixed melting point with a pure sample gives nodepression.)

(f) 60 g. of this hydrazide are converted into the azide using aceticacid and sodium nitrite according to the directions of Example 1 at step(1).

(g) The so obtained dried benzene solution (about 900 cc.), containingthe 5-benzy1oxy-indo1e-3-fl-propionazide, is added dropwise to 76 cc. ofanhydrous benzyl alcohol warmed at 130 C. in anoil bath: continuousdistillation of benzene occurs. The solution is warmed at 130 C. for 30minutes more after complete distillation of benzene. 76 cc. of xyleneare added and the resulting brown solution is evaporated to drynessunder reduced pressure. The residual oil is dissolved in 80 cc. ofbenzene and poured into a column of 1000 g. of aluminium oxide.

After complete elution with benzene the eluted solution is concentratedunder reduced pressure to about 90 00.: addition of 15-20 cc. of hexanecauses the crystallisation of 50.3 g. of 13-[3-(5-benzyloxy)-indolyl]-ethyl carbamic acid benzyl ester, as white needles melting at70-72 C. The mother liquors are again treated with aluminium oxide soobtaining a further crop of the same product. Total yield 60 g. (77% ofthe theoretical amount). (An analytical sample recrystallised frombenzene-hexane melts at 72-73 C. and the determination of nitrogencontent is in good agreement with the formula calc. 7, N found 6.81.)

(h) To a solution of 60 g. of the above benzylurethane in 6 l. ofethanol and 144 cc. of normal hydrochloric acid, 30 g. of palladium oncarbon catalyst are added. The mixture is treated with hydrogen withcontinuous stirring at six atm. pressure (room temperature: about 25 C.)until no more hydrogen is adsorbed.

(i) To the so-obtained ethanolic solution, filtered from the catalyst,containing the S-hydroxy-tryptamine hydrochloride, 34.5 g. of picricacid are added. Elimination of the ethanol under reduced pressure to avolume of 850 cc. causes the crystallisation of most of the5-hydroxytryptamine pierate as red needles: 45.5 g., M.Pt. 185'l8 6 -Byconcentration of the mother liquors further crops of crystals arecollected: the total yield amounted to 60 g. (about 95%). (A mixedmelting point of a water recrystallised sample with pureS-hydroxy-tryptarnine picrate gives no depression.) a

(l) The S-hydroxy-tryptamine may be separated from the alcoholicsolution, after the palladium-hydrogen treatment, in the form of itsdouble salt with creatinine. (sulfate). The handling is as follows: toan ethanolic solution -obtained by treatment of 60 g. of benzylurethanewith hydrogen in presence of 10% palladium on carbon catalyst accordingto the above directions-17 g. of creatinine and then cc. of 2 N sulfuricacid are added. The mixture is heated to boiling with stirring and thencooled to 0 C.; 5 -hydroxy-tryptamine creatinine sulfate separates offas a white crystalline precipitate. The product is filtered on aBiichner funnel, washed with ethanol and dried: yield 55.5 g.: M;Pt.2l0211 C. with dec. (A water recrystallised sample melts at 2l3214 C.with dec. and does not give depression when mixed with anauthenticsample of 5-hydroxy-tryptamine-creatinine-sulfate.)

We have again experienced that, after the benzene solution of the5-benzyloxy-indole-3-p-propionazide (from 60 g. of hydrazide) has beenreacted with benzyl alcohol and all the benzene distilled, the crudebenzyl alcohol solution of the 18-[3-(5-benzyloxy) -indolyl] -ethylcarbamic acid benzyl ester, when diluted to 6 l. with ethanol containing144 cc. of N hydrochloric acid, may be directly submitted topalladium-hydrogen treatment in a perfectly analogous way. In this caseall the benzyl alcohol present is transformed into toluene. Afterelimination of the catalyst from the hydrogenated solution,S-hydroxytryptamine picrate, as well asS-hydroxy-tryptaminecreatinine-sulfate, is obtained in a similar highyield.

What we claim is:

1. The compound 3(S-benzyloxy-Z-carboxyindolyl-3)- propionic acid offormula:

CgHg-CHz-O CHz-CHz-COOH [COOK N H 2. The method of making5(5-benzyloxy-2-carboxyindolyl-3)propionic acid of the formula OOOHwhich comprises boiling w-keto-adipic acid p-benzyloxyphenyl-hydrazoneof the formula CHr-GHr-CHz-C 0 OH N HN in dioxane in the presence ofhydrochloric acid.

References Cited in the file of this patent UNITED STATES PATENTS (Otherreferences on following page)

1. THE COMPOUND B(5-BENZYLOXY-2-CARBOXYINDOLYL-3)PROPIONIC ACID OFFORMULA: